Different biological substances are commonly employed to promote bone growth in medical applications including fracture healing and surgical management of bone disorders including spinal disorders. Spine fusion is often performed by orthopedic surgeons and neurosurgeons alike to address degenerative disc disease and arthritis affecting the lumbar and cervical spine. Historically, autogenous bone grafting, commonly taken from the iliac crest of the patient, has been used to augment fusion between vertebral levels.
One protein that is osteogenic and commonly used to promote spine fusion is recombinant human bone morphogenetic protein-2 (rhBMP-2). Its use has been approved by the US Food and Drug Administration (FDA) for single-level anterior lumbar interbody fusion. The use of rhBMP-2 has increased significantly since this time and indications for its use have expanded to include posterior lumbar spinal fusion as well as cervical spine fusion.
Oxysterols form a large family of oxygenated derivatives of cholesterol that are present in the circulation, and in human and animal tissues. Oxysterols have been found to be present in atherosclerotic lesions and play a role in various physiologic processes, such as cellular differentiation, inflammation, apoptosis, and steroid production. Some naturally occurring oxysterols have robust osteogenic properties and can be used to grow bone. The most potent osteogenic naturally occurring oxysterol, 20(S)-hydroxycholesterol, is both osteogenic and anti-adipogenic when applied to multipotent mesenchymal cells capable of differentiating into osteoblasts and adipocytes.
One such oxysterol is Oxy133 or (3S,5S,6S,8R,9S,10R,13S,14S,17S) 17-((S)-2-hydroxyoctan-2-yl)-10, 13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-3,6-diol, which exhibits the following structures:

Oxy133 is a synthetic small molecule that promotes bone growth in mammals. Currently, the oxysterol class of compounds is analyzed using gas chromatography (GC) with derivatization. This is a lengthy process that requires heating of the samples and is not preferred by regulatory agencies. Currently available industry detection techniques paired with high performance liquid chromatography (HPLC) are not robust or sensitive enough to detect Oxy133 in the presence of related impurities and degradation.
The Oxy133 molecule lacks a chromophore making chromatography insufficient. In addition, known impurities closely related to the parent compound are difficult to detect by techniques such as evaporative light scattering (ELS), refractive index (RI), and mass spectrometry (MS).
Evaluation of purity is required to assure the safety and efficacy of OXY133 and is often achieved by applying an HPLC/UV method. Standard industry detection techniques paired with HPLC are not robust or sensitive enough to detect OXY133 in the presence of related impurities and degradation.
When OXY133 is in a monohydrate form, there is often difficulty analyzing Oxy133 monohydrate due to the presence of related impurities, for example, diastereomers, which need to be separated, quantified and identified. Known impurities are closely related to the parent compound and are difficult to detect by techniques such as evaporative light scattering (ELS), refractive index (RI), and mass spectrometry (MS.)
Therefore, a need to overcome the drawbacks of these detection techniques and to provide reliable analytical methods for the determination of content and purity in samples containing an OXY133 product as part of critical-path activities during the analytical method development (AMD) phase required to validate ICH quality control guidelines. Methods to determine purity in a sample of OXY133 which do not rely on presence of chromophores in the sample would be beneficial. Methods which can detect non-volatile analytes or residues would also be beneficial.